Non-aqueous electrophoresis integrated with electrospray ionization mass spectrometry on a thiol-ene polymer-based microchip device.

Non-aqueous capillary electrophoresis (NACE) on microfluidic chips is still a comparatively little explored area, despite the inherent advantages of this technique and its application potential for, in particular, lipophilic compounds. A main reason is probably the fact that implementation of NACE on microchips largely precluded the use of polymeric substrate materials. Here, we report non-aqueous electrophoresis on a thiol-ene-based microfluidic chip coupled to mass spectrometry via an on-chip ESI interface. Microchips with an integrated ESI emitter were fabricated using a double-molding approach. The durability of thiol-ene, when exposed to different organic solvents, was investigated with respect to swelling and decomposition of the polymer. Thiol-ene exhibited good stability against organic solvents such as methanol, ethanol, N-methylformamide, and formamide, which allows for a wide range of background electrolyte compositions. The integrated ESI emitter provided a stable spray with RSD% of the ESI signal ≤8%. Separation efficiency of the developed microchip electrophoresis system in different non-aqueous buffer solutions was tested with a mixture of several drugs of abuse. Ethanol- and methanol-based buffers provided comparable high theoretical plate numbers (≈ 6.6 × 104-1.6 × 105 m-1) with ethanol exhibiting the best separation efficiency. Direct coupling of non-aqueous electrophoresis to mass spectrometry allowed for fast analysis of hydrophobic compounds in the range of 0.1-5 µg mL-1 and 0.2-10 µg mL-1 and very good sensitivities (LOD ≈ 0.06-0.28 µg mL-1; LOQ ≈ 0.20-0.90 µg mL-1). The novel combination of non-aqueous CE on a microfluidic thiol-ene device and ESI-MS provides a mass-producible and highly versatile system for the analysis of, in particular, lipophilic compounds in a wide range of organic solvents. This offers promising potential for future applications in forensic, clinical, and environmental analysis. Graphical abstract.

Development of a novel sheathless CE-ESI-MS interface via a CO2 laser ablated opening.

A new and easy to construct sheathless capillary electrophoresis electro spray ionization mass spectrometry (CE-ESI-MS) interface was developed that offers several advantages compared to traditional liquid junction interfaces. The fabrication of the device only requires a CO2 laser engraver that most groups working with microfluids have access to. It only takes a few seconds to create a CO2 laser ablated opening in the bare-fused silica capillaries and the opening can be placed as close as a few mm from the spray tip. The capillary is punctured through a silicone tube such that the opening is directly placed inside this tube, which also serves as a liquid reservoir for the make-up liquid. Electrical contact required for both CE separation and ESI is established via the liquid in this reservoir which is in contact with the electrode of an external high voltage power supply. The developed CE-ESI-MS interface is capable of analysing both small molecules and biomolecules such as peptides in physisorbed PEG polymer brush coated capillaries. Proof-of-principle of the interface was demonstrated by analysing a tryptic digest of BSA. Further, a range of drugs of abuse were also investigated. The examined small molecules (pethidine, nortriptyline, methadone, haloperidol and loperamide) have a quantification limit (LOQ) of 150 ng/mL and a detection limit (LOD) of 40 ng/mL (except for loperamide: LOD = 80 ng/mL). Finally, we used our novel CE-MS interface for the analysis of the Aß40 peptide. This is a member of the beta-Amyloid peptide family, involved in the development of Alzheimer's disease. A LOQ of 9 µg/mL was obtained for Aß40, corresponding to 23 fmoles in a sample volume of 11 nL.

A thiol-ene microfluidic device enabling continuous enzymatic digestion and electrophoretic separation as front-end to mass spectrometric peptide analysis.

One of the most attractive aspects of microfluidic chips is their capability of integrating several functional units into one single platform. In particular, enzymatic digestion and chemical separation are important steps in processing samples for many biochemical assays. This study presents the development and application of a free-flow electrophoresis microfluidic chip, and its upstream combination with an enzyme microreactor with immobilized pepsin in the same miniaturized platform. The whole microfluidic chip was fabricated by making use of thiol-ene click chemistry. As a proof of concept, different fluorescent dyes and labeled amino acids were continuously separated in the 2D electrophoretic channel. The protease pepsin was immobilized using a covalent linkage with ascorbic acid onto a high-surface monolithic support, also made of thiol-ene. To show the potential of the microfluidic chip for continuous sample preparation and analysis, an oligopeptide was enzymatically digested, and the resulting fragments were separated and collected in a single step (prior to mass spectrometric detection), without the need of further time-consuming liquid handling steps.

On-chip electromembrane extraction of acidic drugs.

In the present work, a new supported liquid membrane (SLM) has been developed for on-chip electromembrane extraction of acidic drugs combined with HPLC or CE, providing significantly higher stability than those reported up to date. The target analytes are five widely used non-steroidal anti-inflammatory drugs (NSAIDs): ibuprofen (IBU), diclofenac (DIC), naproxen (NAX), ketoprofen (KTP) and salicylic acid (SAL). Two different microchip devices were used, both consisted basically of two poly(methyl methacrylate) (PMMA) plates with individual channels for acceptor and sample solutions, respectively, and a 25 µm thick porous polypropylene membrane impregnated with the organic solvent in between. The SLM consisting of a mixture of 1-undecanol and 2-nitrophenyl octyl ether (NPOE) in a ratio 1:3 was found to be the most suitable liquid membrane for the extraction of these acidic drugs under dynamic conditions. It showed a long-term stability of at least 8 hours, a low system current around 20 µA, and recoveries over 94% for the target analytes. NPOE was included in the SLM to significantly decrease the extraction current compared to pure 1-undecanol, while the extraction properties was almost unaffected. Moreover, it has been successfully applied to the determination of the target analytes in human urine samples, providing high extraction efficiency.

Nanoliter-Scale Electromembrane Extraction and Enrichment in a Microfluidic Chip.

This paper reports for the first time nanoliter-scale electromembrane extraction (nanoliter-scale EME) in a microfluidic device. Six basic drug substances (model analytes) were extracted from 70 µL samples of human whole blood, plasma, or urine through a supported liquid membrane (SLM) of 2-nitrophenyl octyl ether (NPOE) and into 6 nL of 10 mM formic acid as an acceptor solution. A DC potential of 15 V was applied across the SLM and served as the driving force for the extraction. The cathode was located in the acceptor solution. Because of the small area of the SLM (0.06 mm2), the system provided soft extraction with recoveries <1% for the 70 µL samples. Because of the large sample-to-acceptor-volume ratio, analytes were enriched in the acceptor solution. The enrichment capacity was 6-7-fold per minute, and after 60 min of operation, most of the model analytes were enriched by a factor of approximately 400. Because of the SLM and the direction of the applied electrical field, substantial sample cleanup was obtained. The chips were based on thiol-ene polymers, and the soft-lithography-fabrication procedure and the materials were selected in such a way that future mass production should be feasible. The chip-to-chip variability was within 23% RSD (and less than 10% in most cases) with respect to extraction recovery. Our findings have verified that nanoliter-scale EME is highly feasible and provides reliable data, and for future studies, the concept should be tested for applicability in connection with in vitro microphysiological systems, organ-on-a-chip systems, and point-of-care diagnostics. These are potential areas where the combination of soft extraction and high enrichment from limited sample volumes is required for reliable analytical measurements.

UV-vis Imaging of Piroxicam Supersaturation, Precipitation, and Dissolution in a Flow-Through Setup.

Evaluation of drug precipitation is important in order to address challenges regarding low and variable bioavailability of poorly water-soluble drugs, to assess potential risk of patient safety with infusion therapy, and to explore injectable in situ suspension-forming drug delivery systems. Generally, drug precipitation is assessed in vitro through solution concentration analysis methods. Dual-wavelength UV-vis imaging is a novel imaging technique that may provide an opportunity for simultaneously monitoring changes in both solution and solid phases during precipitation. In the present study, a multimodal approach integrating UV-vis imaging, light microscopy, and Raman spectroscopy was developed for characterization of piroxicam supersaturation, precipitation, and dissolution in a flow-through setup. A solution of piroxicam dissolved in 1-methyl-2-pyrrolidinone was injected into a flowing aqueous environment (pH 7.4), causing piroxicam to precipitate. Imaging at 405 and 280 nm monitored piroxicam concentration distributions during precipitation and revealed different supersaturation levels dependent on the initial concentration of the piroxicam solution. The combination with imaging at 525 nm, light microscopy, and Raman spectroscopy measurements demonstrated concentration-dependent precipitation and the formation, growth, and dissolution of individual particles. Results emphasize the importance of the specific hydrodynamic conditions on the piroxicam precipitation. The approach used may facilitate comprehensive understanding of drug precipitation and dissolution processes and may be developed further into a basic tool for formulation screening and development.

Concomitant monitoring of implant formation and drug release of in situ forming poly (lactide-co-glycolide acid) implants in a hydrogel matrix mimicking the subcutis using UV-vis imaging.

For poly (lactide-co-glycolide acid) (PLGA)-based in situ forming implants, the rate of implant formation plays an important role in determining the overall drug release kinetics. Currently, in vitro techniques capable of characterizing the processes of drug release and implant formation at the same time are not available. A hydrogel-based in vitro experimental setup was recently developed requiring only microliter of formulation and forming a closed system potentially suitable for interfacing with various spectroscopic techniques. The aim of the present proof-of-concept study was to investigate the feasibility of concomitant UV imaging, Vis imaging and light microscopy for detailed characterization of the behavior of in situ forming PLGA implants in the hydrogel matrix mimicking the subcutis. The model compounds, piroxicam and α-lactalbumin were added to PLGA-1-methyl-2-pyrrolidinone and PLGA-triacetin solutions. Upon bringing the PLGA-solvent-compound pre-formulation in contact with the hydrogel, Vis imaging and light microscopy were applied to visualize the depot formation and UV imaging was used to quantify drug transport in the hydrogel. As compared to piroxicam, the α-lactalbumin invoked an acceleration of phase separation and an increase of implant size. α-Lactalbumin was released faster from the PLGA-1-methyl-2-pyrrolidinone system than the PLGA-triacetin system opposite to the piroxicam release pattern. A linear relationship between the rate of implant formation and initial compound release within the first 4h was established for the PLGA-NMP systems. This implies that phase separation may be one of the controlling factors in drug release. The rate of implant formation may be an important parameter for predicting and tailoring drug release. The approach combining UV imaging, Vis imaging and light microscopy may facilitate understanding of release processes and holds potential for becoming a useful tool in formulation development of in situ forming implants.

Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a hydrogel-based subcutaneous tissue surrogate and UV-vis imaging.

Phase separation of in situ forming poly (lactide-co-glycolide acid) (PLGA) implants with agarose hydrogels as the provider of nonsolvent (water) mimicking subcutaneous tissue was investigated using a novel UV-vis imaging-based analytical platform. In situ forming implants of PLGA-1-methyl-2-pyrrolidinone and PLGA-triacetin representing fast and slow phase separating systems, respectively, were evaluated using this platform. Upon contact with the agarose hydrogel, the phase separation of the systems was followed by the study of changes in light transmission and absorbance as a function of time and position. For the PLGA-1-methyl-2-pyrrolidinone system, the rate of spatial phase separation was determined and found to decrease with increasing the PLGA concentration from 20% to 40% (w/w). Hydrogels with different agarose concentrations (1% and 10% (w/v)) were prepared for providing the nonsolvent, water, to the in situ forming PLGA implants simulating the injection site environment. The resulting implant morphology depended on the stiffness of hydrogel matrix, indicating that the matrix in which implants are formed is of importance. Overall, the work showed that the UV-vis imaging-based platform with an agarose hydrogel mimicking the subcutaneous tissue holds potential in providing bio-relevant and mechanistic information on the phase separation processes of in situ forming implants.

Automated coating procedures to produce poly(ethylene glycol) brushes in fused-silica capillaries.

Many bioanalytical methods rely on electrophoretic separation of structurally labile and surface active biomolecules such as proteins and peptides. Often poor separation efficiency is due to surface adsorption processes leading to protein denaturation and surface fouling in the separation channel. Flexible and reliable approaches for preventing unwanted protein adsorption in separation science are thus in high demand. We therefore present new coating approaches based on an automated in-capillary surface-initiated atom transfer radical polymerization process (covalent coating) as well as by electrostatically adsorbing a presynthesized polymer leading to functionalized molecular brushes. The electroosmotic flow was measured following each step of the covalent coating procedure providing a detailed characterization and quality control. Both approaches resulted in good fouling resistance against the four model proteins cytochrome c, myoglobin, ovalbumin, and human serum albumin in the pH range 3.4-8.4. Further, even samples containing 10% v/v plasma derived from human blood did not show signs of adsorbing to the coated capillaries. The covalent as well as the electrostatically adsorbed coating were both found to be stable and provided almost complete suppression of the electroosmotic flow in the pH range 3.4-8.4. The coating procedures may easily be integrated in fully automated capillary electrophoresis methodologies.

Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients: Toward a New Approach for Diagnosis and Patient Stratification.

Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.

A simple sheathless CE-MS interface with a sub-micrometer electrical contact fracture for sensitive analysis of peptide and protein samples.

Online coupling of capillary electrophoresis (CE) to electrospray ionization mass spectrometry (MS) has shown considerable potential, however, technical challenges have limited its use. In this study, we have developed a simple and sensitive sheathless CE-MS interface based on the novel concept of forming a sub-micrometer fracture directly in the capillary. The simple interface design allowed the generation of a stable ESI spray capable of ionization at low nanoliter flow-rates (45-90 nL/min) for high sensitivity MS analysis of challenging samples like those containing proteins and peptides. By analysis of a model peptide (leucine enkephalin), a limit of detection (LOD) of 0.045 pmol/µL (corresponding to 67 attomol in a sample volume of ∼15 nL) was obtained. The merit of the CE-MS approach was demonstrated by analysis of bovine serum albumin (BSA) tryptic peptides. A well-resolved separation profile was achieved and comparable sequence coverage was obtained by the CE-MS method (73%) compared to a representative UPLC-MS method (77%). The CE-MS interface was subsequently used to analyse a more complex sample of pharmaceutically relevant human proteins including insulin, tissue factor and α-synuclein. Efficient separation and protein ESI mass spectra of adequate quality could be achieved using only a small amount of sample (30 fmol). In addition, analysis of ubiquitin samples under both native and denatured conditions, indicate that the CE-MS setup can facilitate native MS applications to probe the conformational properties of proteins. Thus, the described CE-MS setup should be useful for a wide range of high-sensitivity applications in protein research.

Flow induced dispersion analysis rapidly quantifies proteins in human plasma samples.

Rapid and sensitive quantification of protein based biomarkers and drugs is a substantial challenge in diagnostics and biopharmaceutical drug development. Current technologies, such as ELISA, are characterized by being slow (hours), requiring relatively large amounts of sample and being subject to cumbersome and expensive assay development. In this work a new approach for quantification based on changes in diffusivity is presented. The apparent diffusivity of an indicator molecule interacting with the protein of interest is determined by Taylor Dispersion Analysis (TDA) in a hydrodynamic flow system. In the presence of the analyte the apparent diffusivity of the indicator changes due to complexation. This change in diffusivity is used to quantify the analyte. This approach, termed Flow Induced Dispersion Analysis (FIDA), is characterized by being fast (minutes), selective (quantification is possible in a blood plasma matrix), fully automated, and being subject to a simple assay development. FIDA is demonstrated for quantification of the protein Human Serum Albumin (HSA) in human plasma as well as for quantification of an antibody against HSA. The sensitivity of the FIDA assay depends on the indicator-analyte dissociation constant which in favourable cases is in the sub-nanomolar to picomolar range for antibody-antigen interactions.

On-chip electromembrane extraction for monitoring drug metabolism in real time by electrospray ionization mass spectrometry.

Sample preparation is an essential step in any bioanalytical procedure and very often the most challenging step in method development. Most of the currently used methods require a relatively large amount of sample and are time consuming. Here, we describe a new approach based on electromembrane extraction (EME) integrated in microfluidic polymer chips. This procedure is fast, requires only small amounts of sample, and may thus be used for monitoring drug metabolism and the formation of metabolites in real time.

Easy peak tracking in CE-UV and CE-UV-ESI-MS by incorporating temperature-correlated mobility scaling.

A simple data reconstruction technique in CE-UV-ESI-MS (where UV stands for ultraviolet) is presented to overcome the drift in mobilities caused by various factors compromising the reproducibility of such data, for example Joule heating effects and the variation in thermostatic control along the capillary, drift in EOF and the suction effect caused by the nebulizing gas in coaxial CE-MS interfaces. We present here a method to transform the traditional time-based electropherogram into the corresponding temperature-correlated mobility scale allowing tracking of analytes independent from capillary dimensions, electric field strengths, temperature control, and distance between the detectors. The main principle of this alignment is based on including the current in the mobility calculations and relating this to the initial electrical resistance of the buffer-filled capillary. The temperature-correlated mobility calculation eliminates the peak shifts due to the viscosity changes, improves the precision of peak identification using the observed temperature-correlated mobilities, and allows a direct comparison of signals from different detection combinations. The method allows peaks from normal CE-UV separations to be correlated with the corresponding peak obtained by MS detection in CE-MS even for differences in capillary dimensions and thermostatic control.

Improving the reproducibility in capillary electrophoresis by incorporating current drift in mobility and peak area calculations.

The traditional way of calculating mobility and peak areas in capillary electrophoresis does not take into account the changes in the buffer viscosity at different thermostatic control and that the analytes may accelerate during the individual runs due to Joule heating effects. We present a method for accounting for these changes based on the monitored changes in current during the separation. The calculation method requires measuring the initial resistance of the buffer filled capillary, performed using a 0.2 min voltage ramping at the start of a separation. The mobility calculation corrected for current drift allowed identification of the tested analytes independent from capillary dimensions, electric field strengths and temperature control. Furthermore, the peak areas become less influenced by the experimental conditions, since the velocities of the analytes passing the detector are corrected for the acceleration during the run. The short voltage ramping could be further used to evaluate the heat transfer of the capillary to the surroundings and to estimate the temperature changes during the separation. The temperature was shown to change the ionization of 2-phenylethylamine in accordance to a pKa dependency of primary amines reported in literature.

Effect of Joule heating on efficiency and performance for microchip-based and capillary-based electrophoretic separation systems: a closer look.

An attempt is made to revisit the main theoretical considerations concerning temperature effects ("Joule heating") in electro-driven separation systems, in particular lab-on-a-chip systems. Measurements of efficiencies in microfabricated devices under different Joule heating conditions are evaluated and compared to both theoretical models and measurements performed on conventional capillary systems. The widely accepted notion that planar microdevices are less susceptible to Joule heating effects is largely confirmed. The heat dissipation from a nonthermostatically controlled glass microdevice was found to be comparable to that from a liquid-cooled-fused silica capillary. Using typically dimensioned glass and glass/silicon microdevices, the experimental results indicate that 5-10 times higher electric field strengths can be applied than on conventional capillaries, before detrimental effects on the separation efficiency occur. The main influence of Joule heating on efficiency is via the establishment of a radial temperature profile across the lumen of the capillary or channel. An overall temperature increase of the buffer solution has only little influence on the quality of the separation. Still, active temperature control (cooling, thermostatting) can help prevent boiling of the buffer and increase the reproducibility of the results.

A microfluidic device with an integrated waveguide beam splitter for velocity measurements of flowing particles by Fourier transformation.

A microfabricated capillary electrophoresis device for velocity measurements of flowing particles is presented. It consists of a 1 x 128 planar waveguide beam splitter monolithically integrated with an electrically insulated fluidic channel network for fluorescence excitation at multiple points. Stray light rejection structures are included in order to suppress unwanted light between the detection regions. The emission pattern of particles passing the detection region was collected by a photomultiplier tube that was placed in close proximity to the channel, thereby avoiding the use of transfer optics. The integrated planar waveguide beam splitter was, furthermore, permanently connected to the light source by a glued-on optical fiber, to achieve a robust and alignment-free operation of the system. The velocity was measured using a Fourier transformation with a Shah function, since the response of the light array was designed to approximate a square profile. Deviations from this response were observed as a result of the multimode nature of the integrated waveguides.

Performance of an in-plane detection cell with integrated waveguides for UV/Vis absorbance measurements on microfluidic separation devices.

A microfluidic device with integrated waveguides and a long path length detection cell for UV/Vis absorbance detection is presented. The 750 microm U-cell detection geometry was evaluated in terms of its optical performance as well as its influence on efficiency for electrophoretic separations in the microdevice. Stray light was found to have a strong effect on both, the sensitivity of the detection and the available linear range. The long path length U-cell showed a 9 times higher sensitivity when compared to a conventional capillary electrophoresis (CE) system with a 75 microm inner diameter (ID) capillary, and a 22 times higher sensitivity than with a 50 microm ID capillary. The linear range was comparable to that achieved in a 75 microm ID capillary and more than twice as large as in a 50 microm ID capillary. The use of the 750 microm U-cell did not contribute significantly to band broadening; however, a clear quantification was made difficult by the convolution of several other band broadening sources.